Abstract：In order to evaluate the safety and physiological function of nicotine metabolic, using the method of biological synthesis preparation of L-6-hydroxy nicotine. we prepared the nicotine metabolic intermediate which is called L-6-hydroxynicotine by Arthrobacter Z3 using nicotine as substrate. We found that by using the analytical HPLC, when the fermentation reacted for 24h, the amount of the target product accumulation reaches the maximum. We take ethyl acetate: methanol: a mixed solution of aqueous ammonia (6:1:0.01) as eluent, the fermented crude product was separated and purity of the target product is 99.00% detected by HPLC. After identified by IR, 1HNMR, 13CNMR and MS, nicotine metabolic intermediate L-6-hydroxynicotine was confirmed.